Project Summary Sepsis, an uncontrolled systemic inflammatory immune response to local infection by bacteria or fungi, is responsible for more deaths than prostate cancer, breast cancer, and AIDS combined, accounts for more than 40% of ICU costs, and is the most expensive inpatient condition in the U.S. (~$20B in annual U.S. healthcare expenditures in 2011). While it has been demonstrated that the time to initiating aggressive treatment is critical to improving outcomes (currently 30-50% mortality) and decreasing costs (>$22,000/case), emergency medicine physicians suffer from a lack of compelling diagnostic tools for detection of sepsis in the emergency department (ED), where >80% of sepsis cases originate in the hospital. CytoVale aims to improve the sepsis treatment paradigm by offering a platform to detect abnormal systemic inflammation at initial presentation. Our diagnostic platform will offer a cost-effective, robust, and rapid sample-to-decision assay (< 10 minutes), in which activated white blood cells that are indicative of uncontrolled systemic inflammation are interrogated in a label-free manner. In order to impact quality of care, the instrument must be situated at the point-of-care (POC), where it can provide rapid results to impact patient care. Currently, this is not feasible, due to manual operations required to prepare samples for the assay (e.g., centrifugation, pipetting) and the lack of an in- sample calibration. Here, we propose to capitalize on the success of our Phase 1 progress of automating the process of sample preparation by now developing a fully integrated module that combines the sample preparation and deformability cytometry microfluidics into a single cartridge and instrument. This will allow for the identification and triaging of patients earlier in their disease process at the ED POC from a finger-prick of blood. Additional cost and patient comfort benefits arise from the ability to track the course of disease and design treatments specific to patient response; saving days spent in the hospital and associated costs. Cytovale also proposes to develop and integrate a deformable sample control. Mechanical standard particles will be used as internal controls within each sample run, to ensure fidelity of the instrument readings and diagnostic accuracy. Not only will this help verify reliability and consistency of experiments, but will also supply a much needed calibration particle to the growing field of cellular biophysics. Combined, these aims will expand accessibility of powerful cell-based assays at the POC.